Isolation And Expression Pattern Of Key Genes Involved In Blue Flower Formation

Commercially important examples are rose, carnation, tulip, and chrysanthemum, which have no blue-flowering varieties. Blue flower breeding also have been one of the hotspots in flower color modification. A main reason for the lack of blue flowers is the absence of 3′,5′-hydroxylated anthocyanins, which are synthesized by a key enzyme, flavonoid 3′,5′-hydroxylase (F3′5′H). Therefore, integration of a F3′5′H gene into such plant species by molecular biological techniques would provide a good opportunity to create novel blue flowers. Anthocaynins biosynthesis is likely to function as multienzyme complexes, and there are at least two enzymes like flavonone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR), competing the intermediates with F3′5′H, so the genes coding these enzymes are also the key member for the blue flower formation.Chrysanthemum is one of the four cut flowers in the world. In China it is so famous and classical that it is of important cultural and economic significance. Blue-flowering is the only type absent from 30,000 chrysanthemum varieties. The phylogenetic relationship between the donor and donee of target gene has been demonstrated to affect the accumulation of target anthocyanins in transformed plants, therefore, the research on the characteristic of blue genes among composite would provide more academic reference for blue chrysanthemum molecular breeding. Cineraria (Pericallis cruentia L′Herit; Senecio cruentus) was chosen as the starting material in this study because it has blue varieties and is a popular potted flower in spring available at local markets in China.Several degenerate primers are designed according to the known F3′5′H amino acid sequence, RT-PCR and RACE are performed to amplified F3′5′H homologous gene form cineraria. The full-length cDNA, named PCFH (GenBank accession no. AY791885), was 1,660 bp and contains an ORF, from 8 bp to 1,522 bp, encoding a polypeptide of 504 amino acid residues. The deduced amino acid sequence had characteristics of P450s. PCFH shared about 45~70% homology with the accessed F3′5′H in GenBank, which proved these related sequences belong to a subfamily of the P450s superfamily, CYP75A.With the same methods, the transcription of F3′5′H gene is also detected in other blue-flowering plants: cornflower (Centaurea cyanus), iris (Iris lactea Pall.) and delphinium (Delphinium grandiflorum L.). DGFH (GenBank accession no. AY856345) is cloned from delphinium, which is 1,693bp, 14～1,522bp is the ORF coding 502aa. The same conservative domain and high sequence similarity...